The transport of all classical neurotransmitters into synaptic vesicles involves a mechanism of H+ exchange and hence depends on a H+ electrochemical driving force (?[unreadable]H+) generated by the vacuolar-type H+-ATPase. However, different transmitters depend to varying extents on the two components of ?[unreadable]H+, the chemical component (?pH) and the electrical component (??), and we currently understand little about the factors that regulate expression of ?[unreadable]H+ as either ?pH or ??. Previous studies have focused almost exclusively on the role of chloride in expression of ?pH, but the carriers responsible and the factors that promote ?? remain poorly understood. In addition, the vesicular glutamate transporters (VGLUTs) show allosteric regulation by chloride and may themselves exhibit a chloride conductance, but the relationship between these properties remains unknown. We also understand little about the mechanism of ionic coupling by the VGLUTs, despite important implications for the presynaptic regulation of quantal size and the activation of glutamate receptors. We will thus 1) Elucidate the factors that drive vesicular glutamate transport by promoting expression of ?[unreadable]H+ as ??. Vesicular glutamate transport depends primarily on ??, and we have recently identified a cation/H+ exchange activity that converts ?pH into ?? and promotes vesicle filling with glutamate. The activity has properties associated with the family of Na+/H+ exchangers and we will now identify the isoform(s) responsible, as well as characterize their role in transmitter release. 2) Characterize the currents associated with VGLUT2. To develop a more robust assay for VGLUT activity, we have expressed an endocytosis-defective mutant of VGLUT2 at the plasma membrane of Xenopus oocytes. We will now use the associated currents to understand the ionic coupling of the VGLUTs and the relationship between different properties ascribed to them, including Na+-dependent phosphate transport, a Cl- conductance and allosteric regulation by Cl-. 3) Identify residues responsible for the differences in ionic coupling by different VGLUT family members. To understand how closely related proteins can mediate transport with different mechanisms of ionic coupling, we will use site-directed mutagenesis of VGLUT2 and a purified bacterial relative, E. coli DgoT. By functional reconstitution of the purified protein, we have found that DgoT mediates H+ cotransport rather than the ??-driven transport characteristic of the VGLUTs, and we will now determine the structural basis for these differences in transport mechanism. PUBLIC HEALTH RELEVANCE: Synaptic transmission involves the regulated exocytosis of vesicles filled with transmitter, but we still understand little about the basic mechanisms that regulate neurotransmitter transport into synaptic vesicles. This program will identify factors that promote the membrane potential driving vesicular glutamate transport. It will also characterize the properties of vesicular glutamate transport through the analysis of associated currents and a bacterial relative. The results will have important implications for our understanding of excitatory neurotransmission, behavior and neuropsychiatric disease.